392
CLAUDE FROMAGEOT AND JACQUES C. SENEZ
preparations are relatively stable and can be maintained for several
weeks at -\-2°, as long as they are kept without air, under hydrogen.
Fischer et al. (175) showed that hydrogenase is inhibited reversibly by
oxygen, regaining full activity when 0 2 is eliminated from the medium,
e.g., by hydrosulfite. On the basis of their observations, they postulated
that hydrogenase, like hemoglobin, might pass reversibly from its normal
form to an oxygenated, inactive form. Irreversible loss of activity, however, results from prolonged exposure to air or addition of oxidants.
3. Nature of the Prosthetic Group
Following the work of Shug et al. (172), hydrogenase has been
considered a molybdoflavoprotein intimately related in structure to the
other metalloflavoproteins and to xanthine oxidase in particular. This
concept is based on various experimental results, notably on the simultaneous presence of molybdenum and flavins in every preparation, regardless of the degree of purification of the hydrogenase. Another important fact is that the hydrogenase of C. pasteurianum, when treated
with ammonium sulfate and dialyzed for a long time, loses a large
part of its activity; this is restored by the addition of both FAD and
molybdenum as molybdic trioxide. The addition of the flavin and the
metal restores the cytochrome c reductase activity (i.e., the reduction
of this cytochrome in an H 2 atmosphere), but not the ability to reduce
methylene blue; this indicates that the reduction of the dye requires a
supplementary factor or electron carrier which is lost during purification.
Other authors have reported data supporting the idea that the metal
involved in hydrogenase activity is ferrous iron. Whiteley and Ordal
(176) showed that the hydrogenase of Micrococcus lactilyticus which
they separated from a xanthine oxidase present in the crude extracts was
inhibited by orthophenanthroline and reactivated by catalytic quantities
of Fe
2+ . Hoberman and Rittenberg (164) concluded that the hydrogenase they were studying was a ferroprotein complex and Waring and
Werkman (177) noted that cells of Aerobacter aerogenes grown on ironfree media did not exhibit hydrogenase activity. Peck and Gest (148)
showed that Fe
2+ activates the hydrogenases of M. lactihfticus and C.
butylicum and concluded that both iron and molybdenum were present
in these enzymes.
Winfield (178) emphasized that an iron requirement or an activation by iron does not necessarily mean that this metal is directly involved in the constitution of the hydrogenase and plays a functional role
in the activation of H 2 . Iron may have only a secondary role of chelation, similar to that observed in other metalloflavoproteins by Mahler
et al. (179). Another possibility is that iron is not involved in hydro-
CLAUDE FROMAGEOT AND JACQUES C. SENEZ
preparations are relatively stable and can be maintained for several
weeks at -\-2°, as long as they are kept without air, under hydrogen.
Fischer et al. (175) showed that hydrogenase is inhibited reversibly by
oxygen, regaining full activity when 0 2 is eliminated from the medium,
e.g., by hydrosulfite. On the basis of their observations, they postulated
that hydrogenase, like hemoglobin, might pass reversibly from its normal
form to an oxygenated, inactive form. Irreversible loss of activity, however, results from prolonged exposure to air or addition of oxidants.
3. Nature of the Prosthetic Group
Following the work of Shug et al. (172), hydrogenase has been
considered a molybdoflavoprotein intimately related in structure to the
other metalloflavoproteins and to xanthine oxidase in particular. This
concept is based on various experimental results, notably on the simultaneous presence of molybdenum and flavins in every preparation, regardless of the degree of purification of the hydrogenase. Another important fact is that the hydrogenase of C. pasteurianum, when treated
with ammonium sulfate and dialyzed for a long time, loses a large
part of its activity; this is restored by the addition of both FAD and
molybdenum as molybdic trioxide. The addition of the flavin and the
metal restores the cytochrome c reductase activity (i.e., the reduction
of this cytochrome in an H 2 atmosphere), but not the ability to reduce
methylene blue; this indicates that the reduction of the dye requires a
supplementary factor or electron carrier which is lost during purification.
Other authors have reported data supporting the idea that the metal
involved in hydrogenase activity is ferrous iron. Whiteley and Ordal
(176) showed that the hydrogenase of Micrococcus lactilyticus which
they separated from a xanthine oxidase present in the crude extracts was
inhibited by orthophenanthroline and reactivated by catalytic quantities
of Fe
2+ . Hoberman and Rittenberg (164) concluded that the hydrogenase they were studying was a ferroprotein complex and Waring and
Werkman (177) noted that cells of Aerobacter aerogenes grown on ironfree media did not exhibit hydrogenase activity. Peck and Gest (148)
showed that Fe
2+ activates the hydrogenases of M. lactihfticus and C.
butylicum and concluded that both iron and molybdenum were present
in these enzymes.
Winfield (178) emphasized that an iron requirement or an activation by iron does not necessarily mean that this metal is directly involved in the constitution of the hydrogenase and plays a functional role
in the activation of H 2 . Iron may have only a secondary role of chelation, similar to that observed in other metalloflavoproteins by Mahler
et al. (179). Another possibility is that iron is not involved in hydro-